We first assessed the effects of anserine on [14C]glycylsarcosine [14C]Gly-Sar uptake into Caco-2 cells expressing human PEPT1 and into spontaneous hyperten-sive rat kidney proximal tubul
Trang 1proton-coupled peptide transporters
Stefanie Geissler1, Madlen Zwarg1, Ilka Knu¨tter1, Fritz Markwardt2and Matthias Brandsch1
1 Membrane Transport Group, Biozentrum of Martin-Luther-University Halle-Wittenberg, Halle, Germany
2 Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle-Wittenberg, Halle, Germany
Introduction
The bioactive dipeptide anserine
(b-alanyl-1-N-methyl-l-histidine) is found in considerable amounts in
skele-tal muscle and brain of vertebrates [1,2] It is formed
as a secondary product through the methylation of the
dipeptide carnosine (b-alanyl-l-histidine) [3] The
reac-tion is catalysed by carnosine N-methyltransferase
[3,4] Both anserine and carnosine exert antioxidative
properties, pH buffering capacity and transglycating
activity [5,6] Anserine and carnosine are thought to
inhibit lipid oxidation by a combination of free radical
scavenging and metal chelation [7] Furthermore,
anserine and carnosine enhance postdenervation depolarization by the inhibition of NO production [8] Because of the many recent reports on the endoge-nous biochemical effects of anserine on the one hand, and its presence in human diet on the other, the intestinal absorption of anserine has received increas-ing interest recently In 2009, the intestinal absorption
of anserine after the ingestion of an anserine-contain-ing diet, and its blood clearance, were studied [2] Ingested anserine is absorbed intact into human blood and is then hydrolysed to p-methyl-l-histidine and
Keywords
carnosine; intestine; kidney; PEPT1; PEPT2
Correspondence
M Brandsch, Membrane Transport Group,
Biozentrum of Martin-Luther-University
Halle-Wittenberg, Weinbergweg 22,
D-06120 Halle, Germany
Fax: +49 345 5527258
Tel: +49 345 5521630
E-mail: matthias.brandsch@biozentrum.
uni-halle.de
(Received 4 November 2009, revised 2
December 2009, accepted 2 December
2009)
doi:10.1111/j.1742-4658.2009.07528.x
The bioactive dipeptide derivative anserine (b-alanyl-1-N-methyl-l-histidine)
is absorbed from the human diet in intact form at the intestinal epithelium The purpose of this study was to investigate whether anserine is a substrate
of the H+⁄ peptide cotransporters 1 and 2 (PEPT1 and PEPT2) We first assessed the effects of anserine on [14C]glycylsarcosine ([14C]Gly-Sar) uptake into Caco-2 cells expressing human PEPT1 and into spontaneous hyperten-sive rat kidney proximal tubule (SKPT) cells expressing rat PEPT2 Anser-ine inhibited [14C]Gly-Sar uptake with Ki values of 1.55 mm (Caco-2) and 0.033 mm (SKPT) In HeLa cells transfected with pcDNA3-hPEPT1 or pcDNA3-hPEPT2, Kivalues of 0.65 mm (hPEPT1) and 0.18 mm (hPEPT2) were obtained We conclude from these data that anserine is recognized by PEPT1 and PEPT2 Carnosine also inhibited [14C]Gly-Sar uptake Using the two-electrode, voltage-clamp technique at Xenopus laevis oocytes, strong hPEPT1-specific inward transport currents were recorded for Gly-Sar, anserine and carnosine, but not for glycine We conclude that anserine and carnosine interact with the human intestinal peptide transporter and are transported by hPEPT1 in an active, electrogenic H+symport As PEPT1
is the predominant transport system for di- and tripeptides at the intestinal epithelium, this transporter is most probably responsible for the intestinal absorption of anserine after food intake In addition, anserine might be useful for the design of new substrates of peptide transporters, such as prodrugs, that can be administered orally
Abbreviations
Gly-Sar, glycylsarcosine; hPEPT, human PEPT; PEPT1, H+⁄ peptide cotransporter 1; PEPT2, H + ⁄ peptide cotransporter 2; rPEPT, rat PEPT; SKPT, spontaneous hypertensive rat kidney proximal tubule.
Trang 2b-alanine by serum and tissue carnosinases According
to the authors, this was the first study to demonstrate
the intestinal absorption of anserine [2] It should be
noted, however, that, as early as 1976, Hama et al [9]
concluded from their studies on the absorption of
b-alanine, anserine and carnosine that physiological
amounts of anserine (and carnosine) are absorbed
from rat small intestine in intact form Very recently,
Yeum et al [10] have investigated the metabolic
stabil-ity of carnosine and anserine in human serum and
their absorption kinetics in vivo Again, the anserine
concentration was increased significantly after
diges-tion of anserine-rich food The molecular mechanism
of anserine uptake remains unknown Based on the in
vivo data and molecular structure of anserine, we
hypothesized that anserine might be recognized by the
intestinal peptide transporter At the intestinal
epithe-lium, di- and tripeptides are transported from the
lumen into the enterocytes by H+⁄ peptide
cotransport-er 1 (PEPT1) (peptide transportcotransport-er 1) (for a review, see
[11,12]) At the renal epithelium, small peptides are
reabsorbed from the glomerular filtrate into the cells
by the subtypes PEPT1 and PEPT2 PEPT2 is also
expressed in other tissues, such as lung and choroid
plexus In addition to peptides, both PEPT1 and
PEPT2 also accept several pharmacologically relevant
peptidomimetics as substrates, such as many b-lactam
antibiotics, valacyclovir and d-aminolaevulinic acid
[12] The intestinal proton-coupled peptide transport
system also accepts carnosine as substrate [13,14] (for
a review, see [15]) To the best of our knowledge, the
transport of anserine by H+⁄ peptide cotransporters
has not yet been studied Interaction with these
carri-ers would not only deliver new information on the
substrate specificity of the transporters, but transport
by PEPT1 would also explain the high oral availability
of anserine
Results and discussion
Inhibition of [14C]glycylsarcosine ([14C]Gly-Sar)
uptake at Caco-2 and spontaneous hypertensive
rat kidney proximal tubule (SKPT) cells by
anserine
Caco-2 and SKPT cell cultures are well-established
systems for intestinal and renal peptide transport studies
Caco-2 cells express the human low-affinity,
high-capacity (‘intestinal’)-type peptide transport system
PEPT1, whereas SKPT cells express the rat high-affinity,
low-capacity (‘renal’)-type system PEPT2, but not
PEPT1 [16–18] In the present investigation, we first
determined the effect of anserine on [14C]Gly-Sar uptake
Gly-Sar is used as reference substrate for peptide transport studies because of its relatively high enzymatic stability At concentrations of 10 mm (Caco-2) and 2 mm (SKPT), anserine strongly inhibits the uptake of [14C]Gly-Sar (10 lm) by 76% and 79%, respectively With both cell lines, competition assays at increasing concentrations of Gly-Sar and anserine were performed From the inhibition curves shown in Fig 1, IC50values, i.e the inhibitor concentration necessary to inhibit carrier-mediated [14C]Gly-Sar uptake by 50%, were calculated and converted into Ki values, as described previously [16–19] Gly-Sar, a prototype substrate for PEPT1 and PEPT2, displayed Ki values of 0.74 ± 0.01 mm and 0.11 ± 0.01 mm, respectively (Fig 1, Table 1) Anserine inhibited [14C]Gly-Sar uptake mediated by PEPT1 into Caco-2 cells with a Ki value
of 1.55 ± 0.02 mm The Ki value of anserine for the inhibition of [14C]Gly-Sar uptake via PEPT2 into SKPT cells was 0.033 ± 0.001 mm (Table 1)
The apparent affinity of anserine is thereby lower than that of Gly-Sar at PEPT1, but higher at PEPT2
As reviewed earlier, most dipeptides composed of natural amino acids display Ki values in the range 0.07–0.7 mm at PEPT1 and 5–100 lm at PEPT2 [11,12] According to our classification [12], anserine can be considered as a medium-affinity ligand for human PEPT1 and a high-affinity ligand for rat PEPT2
Effect of anserine on [14C]Gly-Sar uptake in HeLa-hPEPT1 and HeLa-hPEPT2 cells Caco-2 and SKPT cells originate from different species, man and rat, respectively To rule out the
pos-Fig 1 Inhibition of [ 14 C]Gly-Sar uptake into Caco-2 and SKPT cells
by anserine Uptake of 10 l M [ 14 C]Gly-Sar was measured for
10 min at pH 6.0 in the presence of increasing concentrations of anserine (n = 3–4).
Trang 3sibility that differences in substrate recognition
between hPEPT1 and rPEPT2 reflect species’
differ-ences and to confirm the affinity constants obtained in
Caco-2 and SKPT cells in a second, independent
approach, we performed transport studies with cloned
human PEPT1 and PEPT2 [17] The interaction of
anserine and, for comparison, carnosine and Gly-Sar
with hPEPT1 and hPEPT2 was studied in competition
assays after heterologous expression of the transporters
in HeLa cells (Fig 2) For anserine, Ki values of
0.65 ± 0.02 mm and 0.18 ± 0.01 mm were determined
at hPEPT1 and hPEPT2, respectively (Table 1)
Carnosine inhibited [14C]Gly-Sar uptake with Kivalues
of 1.7 ± 0.1 mm (hPEPT1) and 0.06 ± 0.01 mm
(hPEPT2) Unlabelled Gly-Sar inhibited [14C]Gly-Sar
uptake with Ki values of 0.64 ± 0.02 mm (hPEPT1)
and 0.24 ± 0.02 mm (hPEPT2) These results clearly
show that anserine interacts specifically with hPEPT1
and hPEPT2 and that the compound inhibits the
uptake of the prototype substrate Gly-Sar
Transport of anserine by hPEPT1 expressed in Xenopus laevis oocytes
Inhibition of [14C]Gly-Sar uptake at native intestinal or renal cells, or at transfected cells expressing peptide transporters heterologously, does not allow the conclu-sion to be drawn that the inhibiting, competing com-pound – in this case anserine – is indeed transported Anserine could represent an inhibitor blocking directly the binding site of the carrier Alternatively, the results obtained so far do not rule out an indirect effect, for example an effect on the proton gradient, as the driving force of [14C]Gly-Sar uptake Employing the two-electrode, voltage-clamp technique, we therefore inves-tigated whether anserine is able to generate currents at
X laevis oocytes expressing hPEPT1 These currents occur when a compound is cotransported by PEPT1 with H+ in an electrogenic manner As shown in Fig 3, anserine (10 mm) generated inward currents (1254 ± 44 nA) comparable with those generated by
Table 1 Inhibition constants (K i ) of Gly-Sar, anserine and carnosine at PEPT1 and PEPT2 Uptake of [ 14 C]Gly-Sar in Caco-2 and SKPT cells,
or in HeLa cells transfected with hPEPT1- or hPEPT2-cDNA, was measured at pH 6.0 for 10 min at increasing concentrations of unlabelled dipeptides Kivalues were derived from the competition curves shown in Figs 1 and 2 (n = 4) ND, not determined.
Ki(m M )
Compound
hPEPT1 Caco-2
rPEPT2 SKPT
hPEPT1 HeLa
hPEPT2 HeLa
Fig 2 Inhibition of [14C]Gly-Sar uptake into HeLa cells transfected
with pcDNA3-hPEPT1 and pcDNA3-hPEPT2 constructs by anserine,
carnosine and Gly-Sar Uptake of 20 l M [ 14 C]Gly-Sar was measured
for 10 min at pH 6.0 in the presence of increasing concentrations
of the compounds for the determination of IC50values (n = 3–4).
10 sec Fig 3 Electrophysiological analysis of anserine transport in hPEPT1-cRNA-injected X laevis oocytes (membrane potential, )60 mV; pH 6.5) Lower trace: currents induced by 10 m M anser-ine, carnosine and Gly-Sar, and 20 m M glycine Upper trace: mea-surement in water-injected oocytes.
Trang 4the prototype transporter substrate Gly-Sar
(1220 ± 42 nA) and by the structurally related
sub-strate carnosine (1075 ± 24 nA) No currents were
observed for either of the test compounds in
water-injected oocytes (Fig 3) Hence, indirect
PEPT1-independent effects of anserine can be ruled out
PEPT1 does not accept free amino acids as substrates
Therefore, glycine was used as negative control in these
experiments No inward currents could be observed
(Fig 3)
We conclude from these data that anserine is
recog-nized by the proton-coupled peptide transporters
PEPT1 and PEPT2 with medium affinity Anserine is
able to displace other substrates from the transport
process The experiments show that anserine and
car-nosine are transported by hPEPT1 in an active,
elec-trogenic manner by an H+ symport As PEPT1 is the
predominant transport system for di- and tripeptides
at the intestinal epithelium, this transporter is most
probably responsible for the intestinal absorption of
anserine after food intake After entering the blood
compartments and tissues, the hydrolysis of anserine –
which is relatively resistant against intestinal
dipeptid-ases – occurs in serum caused by the activity of
carno-sinases [10,20,21]
With regard to the structural requirements for
PEPT1 and PEPT2 substrates, it is surprising that
anserine, with its N-terminal b-amino acid, displays
such high affinity Therefore, in addition to the
physio-logical and biochemical aspects of anserine transport,
this compound might also be useful for the design of
new substrates of peptide transporters, such as
pro-drugs, that can be administered orally
Experimental procedures
Materials
Caco-2 and HeLa cells were obtained from the German
Collection of Microorganisms and Cell Cultures
(Braun-schweig, Germany) The renal cell line SKPT-0193 Cl.2,
established from isolated cells of rat proximal tubules, was
provided by U Hopfer (Case Western Reserve University,
Cleveland, OH, USA) [16] Cell culture media, supplements
and trypsin solution were purchased from Life
Austria) Fetal bovine serum was obtained from Biochrom
Healthcare (Little Chalfont, Buckinghamshire, UK)
Anser-ine was purchased from Bachem (Weil am Rhein,
Ger-many), and Gly-Sar and carnosine from Sigma-Aldrich
(Deisenhofen, Germany)
Culture of Caco-2 and SKPT cells
with minimum essential medium supplemented with 10%
nones-sential amino acid solution [16–18] Subconfluent cultures (80% of confluence) were treated for 5 min with Dulbecco’s phosphate-buffered saline, followed by a 2 min incubation with trypsin solution For uptake experiments, cells were seeded in 35 mm disposable Petri dishes (Sarstedt,
monolayers reached confluence the next day The uptake measurements were performed on the seventh day after seed-ing The protein content per dish was determined using a
Bonn, Germany) according to the manufacturer’s protocol The culture medium for SKPT cells was Dulbecco’s
supplemented with fetal bovine serum (10%), gentamicin
cells per dish The uptake measurements were performed on the fourth day after seeding [16–18]
Heterologous expression of human PEPT1 and human PEPT2 in HeLa cells
HeLa cells were routinely cultured with Dulbecco’s modi-fied Eagle’s medium with Glutamax, supplemented with
cDNA of human PEPT1 and PEPT2 was cloned into pcDNA3 using the pBluescript constructs as a template for PCR, and XhoI and BamHI as restriction sites [17] The
sequencing Human PEPT1 and human PEPT2 were heter-ologously expressed in HeLa cells using pcDNA3-hPEPT1
or pcDNA3-hPEPT2 constructs (1 lg per well) and Turbo-fect (1.5 lL per well; Fermentas, St Leon-Rot, Germany), according to the manufacturer’s protocol Transfection was performed 1 h postseeding in 24-well plates [17]
[14C]Gly-Sar uptake measurements
cells cultured on plastic dishes was measured at room tem-perature, as described previously [16–18] The uptake buffer
carnosine (0–10 mm, pH readjusted if necessary) After incubation for 10 min, the monolayers were quickly washed four times with ice-cold uptake buffer, solubilized and
Trang 5pre-pared for liquid scintillation spectrometry [16–18] The
or 20 mm (SKPT) of unlabelled Gly-Sar, represented 8.0%
and 12.3% of total uptake, respectively This value was
taken into account during nonlinear regression analysis of
and HeLa-hPEPT2 cells grown in 24-well plates was
performed 20–24 h post-transfection at room temperature in
the same manner, except that the tracer concentration was
20 lm
Construction of pNKS-hPEPT1 and in vitro cRNA
synthesis
The X laevis oocyte expression vector pNKS was kindly
provided by Professor G Schmalzing (RWTH, Aachen,
Germany) This vector contains the 5¢ and 3¢ UTRs of the
cDNA into pNKS, AatII and XbaI restriction sites were
introduced at the 5¢ and 3¢ ends, respectively, by PCR As
template, the pBluescript-hPEPT1 vector was used After
restriction enzyme digestion, the PCR product was ligated
into the digested pNKS vector The insertion of the correct
cDNA was verified by sequencing The pNKS-hPEPT1
construct served as template for cRNA synthesis After
linearizing the plasmids with NotI, cRNAs were synthesized
Huntingdon, Cambridgeshire, UK) The cRNAs were
purified with the MEGAclear kit (Ambion), and the
concentration was determined by UV absorbance at
Xenopus laevis oocytes expressing hPEPT1 and
electrophysiology
Oocytes were surgically removed from anaesthetized X laevis
frogs, dissected and defolliculated as described by Riedel
sulfo-nate (Sigma-Aldrich) was used The removed oocytes were
Healthy-looking oocytes (stages V–VI) were manually
hPEPT1 were injected per oocyte Water-injected oocytes
were used as controls Injected oocytes were maintained at
Five days postinjection, electrophysiological measurements
were performed Oocytes were placed in a flow-through
absence or presence of anserine and carnosine at a concentra-tion of 10 mm Quick and reproducible soluconcentra-tion exchanges were achieved using a small tube-like chamber (0.1 mL) combined with fast superfusion [22–25] Microelectrodes with resistances between 0.8 and 1.4 MX were made of borosili-cate glass and filled with 3 m KCl Whole-cell currents were recorded and filtered at 100 Hz using a two-electrode, voltage-clamp amplifier (OC-725C, Hamden, USA) and sampled at 85 Hz Oocytes were voltage clamped at a
Data analysis
Experiments were performed in duplicate or triplicate, and each experiment was repeated two to three times Results are given as the means ± standard errors The concentra-tion of the unlabelled compound necessary to inhibit
deter-mined by nonlinear regression using the logistical equation
the initial Y value, Min is the final Y value and the power
developed by Cheng and Prusoff [19]
Oocyte data were analysed using the superpatch 2000 program (Julius-Bernstein-Institute of Physiology, SP-Ana-lyzer by T Bo¨hm, Halle, Germany) The statistical values
of the oocyte experiments were taken from the measure-ments of three to seven oocytes each from two batches of oocyte preparation Currents induced by the application
of anserine and carnosine were calculated as the difference
in the currents measured in the presence and absence of substrate
Acknowledgements
This study was supported by Deutsche Forschungs-gemeinschaft grant BR 2430⁄ 2-1 and by the State Sax-ony-Anhalt Life Sciences Excellence Initiative Grant
#XB3599HP⁄ 0105T The authors thank Monika Schmidt for excellent technical assistance
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